CS249311B1 - Proteolytic wound dressing - Google Patents

Abstract

A powder, which serves for covering and treatment of ulcerous and necrotic wounds consists of porous spherical cellulose with particle size 0.05 to 0.5 mm, which contains an immobilized protease e.g. chymotrypsine, trypsin or subtilisine.

Description

The invention relates to a proteolytic wound cover in the form of a backfill which serves to cover and treat purulent and necrotic wounds.

In conventional pharmaceutical practice, powders consisting of various bases (starch, talc, etc.) and bactericides or bacteriostatic active substances, in particular antibiotics, sulfonamides and antifungals, are recommended for this purpose.

Recently, wound-absorbing covers in the form of powders which contain a hydrophilic polymer as the main component have proved to be useful in the treatment of wounds. Their effect is to remove fluids from the wound surface by sorption in the porous structure of the active polymer component and by suction by capillary forces into the interparticle spaces of the backfill layer. In this way the exudate is removed from the wound, but also bacteria, substances causing inflammatory processes and possibly toxins are removed.

In particular, cross-linked dextran preparations have penetrated medical practice (B. S. Jacobsson et al., Scand. J. Plast. Reconstr. Surg. 10, 65-72 (1976)). Promising results have also been achieved with cellulose-based polymer compositions containing in admixture a highly hydrophilic polymeric component such as carboxymethylcellulose (H. Dautzenberg et al., Absorbent Wound Cover, PV 4129-82).

In the further development of dusting powders for the treatment of festering wounds, attention is paid to cellulose powders containing immobilized enzymes of the type proteas 1981). In this case, enzymatic action is used to achieve the cleaning effects.

Proteolytic wound covers cause hydrolytic decomposition and dissolution of necrotic tissues and pus in the wound surface, thereby simultaneously removing the environment for bacterial growth and interrupting the suction of toxic products from the wound.

Practical use of the proteolytic principle presupposes solving a number of problems in the preparation of an effective cover that would not cause adverse side effects. When used in human medicine, this is particularly the prevention of the entry of cellulose or possibly derivatized cellulose into the bloodstream, since the human body does not have enzymatic systems to remove such substances. However, the present solutions for proteolytic cover preparation assume only the use of conventional celluloses, ie ground ion exchange powders with a fibrous structure, containing fine dust fractions that may enter the blood. ”

The proteolytic principle should not be used without the simultaneous application of the previously known absorption principle in order to achieve maximum efficiency of the covers. In the prior art, however, only conventional cellulose with high crystallinity, low porosity, i.e. providing covers with relatively low absorbency, appeared as starting material.

In addition, using the proteolytic principle, it is desirable to search for methods of immobilizing proteases in cellulose that preclude the release of the enzyme from the matrix and the penetration of its soluble form into the bloodstream, causing an allergic reaction as an antigen.

The current disadvantages in using the proteolytic principle are solved by the novel wound cover according to the invention.

The subject of the invention is a proteolytic wound cover characterized in that it consists of spherical particles with a diameter of 0.05-0.5 mm, preferably 0.1-0.3 mm, based on derivatised cellulose with immobilized enzymes of the chymotrypsin family, trypsin , subtilisin. Another object of the invention is a process for the production of a pearl cellulose wound proteolytic wound cover prepared by the suspension process of U.S. Pat. AO 172 640, characterized in that pearl cellulose swollen in water and never dried, with a particle size of 0.07-0.7 mm, preferably 0.14-0.4 mm, is completely freed of toxic, washing or distillation with water vapor impurities, activated for enzyme binding, modified by immobilization of chymotrypsin, trypsin, succinic acid proteases, alternately washed with pH 8.5-5.5 buffer and pH 4-5 pH buffer of zero wash liquor, and then washed with a buffer of pH 7.5 to 85 and dried to 0.1% to 15% of the residual water content of the dry matter, preferably by lyophilization.

The regular spherical form of the individual particles constituting the new wound cover, together with the selected particle size and particle size distribution (0.05 mm - 0.5 mm, preferably 0.1 - 0.3 mm), ensures easy maaipploovaelnost in production and In use, a smooth flow of powder upon the wound spreading and in particular satisfies the requirement to prevent cellulose from entering the bloodstream.

The production of the new cover is preferably based on regenerated spherical cellulose prepared in accordance with U.S. Pat. AO 172 640. Its forefoot is of high porosity, facilitating activation for enzyme binding as well as self immobilization of proteases.

The porous hybrids character of the bead cellulose carrier is retained even after activation, stabilization and drying. Freeze-drying has been found to be a drying method, which removes water gently with respect to the bound enzyme and provides a dry cellulosic derivative with sufficient absorption moiety.

Pearl cellulose prepared as described in U.S. Pat. AO 172 640 is completely free of soluble constituents, in particular of all impurities with the toxic effects by which it has been contaminated during preparation (xanthogenate decomposition products, dispersion medium residues such as chlorobenzene). For this purpose, it is washed with water at 50-90 ° C, with ethanol or both. Chlorobenzene is also removed particularly efficiently by exaggerating with water vapor.

A variety of methods known in the literature can be used to activate cellulose for the binding of proteases (Handbook of Enzyme Biotechnology, edited by A. Wiseman, E. Horwood Limii., CCichester, 1975), and one that provides a sufficiently stable binding between the enzyme and the cellulose and not the? utam? product by toxic substances which would act during the application of the cover, for example by being released or acting by direct contact.

Suitably, the periodate oxidation is relatively simple, easy to carry out, and provides products of sufficient vigor, exhibiting a high absorption effect.

A variety of proteases are suitable for ioobiiisations, esp. trypsin, го ^^ рз ^, subitlitii, but also thsroobysin, papain etc. Binding of the enzyme to cellulose activated by iodisasone oxidation, ie containing reactive aldehyde groups, is carried out in two stages: First, cellulose compounds with Schiff base type enzyme are formed. and these are stabilized by reducing unreacted aldehyde groups with sodium borohydride.

An important step in the preparation of the novel type of cover is the perfect removal of soluble protease-containing fractions from the treated cellulose beads. This is weak with repeated shyness. alkaline (pH 8.5-9.5) and weakly acidic (pH 4-5) buffers in static and column design.

For example, 0.1 M acetate buffer containing 1 M NaCl (pH 5.5) or both NaCl-free buffers, which are applied to the cellulose product after β-up to zero proteolytic activity of the liquid phase, are suitable for this purpose. The alternation of lower and higher pH buffers of variable ionic strength facilitates the elution of the covalently unbound protein by sluggish suppression of electrolyte and hydrophobic interactions.

In order to obtain a perfectly stable preparation with a purified enzyme, the beads are finally washed with borate buffer, for example pH 8, and lyophilized in suspension with this buffer.

Under such conditions, a product is obtained containing only the covalently bound enzyme and no soluble fraction thereof that could penetrate the blood and become an antigen when treating wounds.

A proteolytic cover with chymotrypsin immobilized was tested in clinical practice. Not only did it show prettetlytic activity on necrotic tissue, pus and fibrin, but it did not harm healthy tissue at all, and in accordance with its ability and porous structure of the cellulosic matrix, it excelled in its high absorption capacity.

It aspirated wound exudate containing cleaved tissue necroses and bacteria. Binding of the enzyme to the carrier prevented its autolysis and thereby lost its activity during the action. Prolonged proteolytic activity of chymoorypsin resulted in a large-scale release of necrosis from the wound and its gradual clearing.

The morning infected secretion was actively absorbed by the particulate backfill, thereby preventing the maLacTac edges of the wound and measuring the medium for growth of the bacterial infection. The original set of both principles, both glythiocyte and absorption, achieved rapid cleansing of infectious necrotic defects, formation of clean granulations and rapid wound healing.

It has been found that the new cover is suitable for purulent or necrotic wounds of all kinds, including surgical wounds healing per secundam, abscesses, coated and non-healing ulcers of various origins (leg, x-ray, trophic ulcers), sphincter necrosis defects, infected and necrotic defects after acral thrombocytes, indian and necrectomies of gangrene as well as gangrene of trtertsklertic origin, carbunkles, burns II. and III. grade, infected open fractures, treatment of stubble stumps, disintegrated and festering tumors. Many harmful effects were not observed when you felt a new cover.

In the following, the method of manufacturing the covers according to the invention and its use is explained in more detail, but not by way of example, by way of example.

Example 1

Preparation of capsule with bound cytotoxin

a) Introduction of the aldehyde groups by oxidation with sodium perchlorate

Preference was given to pearl cellulose prepared according to US. and 172 640, not dried, swollen in water, with a P = 90% (total pore content by volume) cellulose was completely free of impurities by washing with hot water (90 ° C, 5 h, 20-fold excess) and distillation with water 1000 g of aspirated cellulose was suspended in 5 liters of 0.1 M ice and stirred at room temperature for 45 minutes, after the oxidation was complete, the oxidized cellulose was washed with about 20 liters of diatomaceous earth for a period of time. The reaction mixture was then transferred to a column and washed with water until the conductivity was equal to that of the distilled water (over ooc).

b) Binding of cymotrypsin to oxidized cellulose

000 g of washed oxidized cellulose was suspended in 1 liter of 0.1 M borate buffer (pH 9) containing 5 g of cymotrypsin (the proteolytic activity of the solution was determined to be 5.35 µm / ml using a denatured Ueootgobin solution, pH 8). Suspension ^A 280 was stirred at room temperature. The binding rate was monitored based on a decrease in protein activity in the binding solution. After 1 hour, when the gentle activity dropped to 0.1 ml / ml, the binding was stopped by aspirating the binding solution.

280

The rate of chymotrypsin binding increases with increasing pH. At the same time, the solubilization of cellulose also increases, depending on the oxidation time. The chosen procedure is based on a series of comparative experiments and is a compromise between the amount of bound enzyme and cellulose losses due to solubilization.

c) Reduction of chyootrypsine-coupled cellulose to stabilize the Schiff bond between the support and enzyme and removal of unreacted aldehyde groups

The chymoorypsin-bound cellulose was suspended in 1 liter of 0.1 M borate buffer (pH 9) in which 500 mg of NaBH4 was dissolved. After stirring at room temperature for 20 minutes, the reduction was stopped by aspirating the reducing solution, and the reduction was repeated once more in the same manner with a new sodium borohydride solution. The reduction was performed by adding NaBH4 solution at a lower concentration for two times to prevent loss of proteolytic activity of chymmorypsin by possible reduction of disulfide bridges.

d) Washing and Lyophilization of Cellulose with Chymoorypsin Bound

After completion of the reduction, chymmorypsin-bound cellulose was washed alternately with 2 liters of 0.1 M borate buffer containing 1 M NaCl (pH 9), 2 liters of 0.1 M acetate buffer containing 1 M NaCl (pH 4.5), followed by the same volumes of both buffers without NaCC. Thereafter, the hhymotrypsin-coupled cellulose was transferred to a column, where the washing with all of the above buffers was repeated until zero effluent activity was observed.

Finally, chymoorypsin-bound cellulose was washed with 0.25 M borate buffer pH 8 and was also filtered in suspension with this buffer. In this way, a preparation was obtained which contained 5 mg of active chymoorypsin per g of dry cellulose. Comparative amino acid analysis of the prepared sample and the sample additionally washed with 6 M guanidine hydrochloride and desiccated water showed that all chyimorypsin is covalently bound to cellulose and therefore cannot pass into the blood and become an antigen when applied.

Example 2

Preparation of the capsule with covalently coupled trypsin α-α-trypsin was performed in a sterile manner as in case 1. A preparation was obtained which contained 8.2 mg of active trypsin per g of filtered cellulose. Due to its narrower specificity (it breaks down proteins only behind the basic amino acids lysine and arginone), trypsin breaks down proteins at fewer sites.

Example 3

Preparation of cover with covalently attached subtiliijneo

The bacterial phase immunization was carried out in the same manner as in Example 1. A preparation was obtained which contained 11.3 mg of active enzyme per 1 g of the sterilized preparation.

Example 4

The protective cover with hhymotrypsin bound according to example 1 was applied in 7 patients for 1 to 4 weeks, in 6 cases with very good effect manifested by suppression of suppurative secretion, cleansing of the defect bottom and release of adherent necroses, which were surgically larger removed by necrectomy.

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Before the next application, the defect was flushed with hydrogen peroxide and 2 per mille chloramine solution. This procedure resulted in vivid red granulation and epithelialization of the defects from the edges with subsequent reduction. After the end of treatment, Panthenol spray was applied topically to the epithelial defects.

In 1 case the suppurative secretion decreased, 1/5 of the defect was cleaned into live granulations, but in the other part of the defect deeply affecting adhering necroses in the field of chronic ischemia caused by a combination of diabetic microangiopathy and obliterating arteriosclerosis of peripheral arteries. weaving. After withdrawal of the preparation after 4 weeks, the defect progressed in the sense of absent diabetic phlegmones.

Examples of use:

1) Man, 72 years old, 3 cm diameter defect after amputation of the 4th toe of the right foot for diabetic gangrene 8 wet necroses on the bottom, with suppurative secretion - after 2 weeks of application the product is mostly pure granulation with isolated adhering necroses in the media edge of the wound, without purulent secretion.

2) Male, 60 years old, after amputation of right lower leg for diabetic gangrene, where a defect of 5 cm in diameter, 2.5 cm deep, with necroses at the bottom in the lateral part of the amputation stump developed after absent phlegmon media-coated granulations of 10 cm in length and 1 cm in width, with purulent secretion. After 4 weeks of application, almost complete healing was achieved except for a defect of 2 cm diameter and 1 cm depth in the lateral part of the stump, with clean granulations and continued epithelization from the edges.

3) Man, age 66, defect after amputation I.-III. finger including the heads of appropriate metatars for diabetic gangrene, size 4-5 cm, partly with coated granulations, partly 8 adhering necroses, in the lateral part defect depth up to 1 cm. After 4 weeks of application, clean granulation was achieved over the entire defect range with continued epithelialization from the edges and reduction to 3 cm in diameter.

- The same patient, 66 years old, male, who needed distal half of gangrene

4. Finger Amputation including Head IV. metatarsus, a defect with a diameter of 3 cm, with a covered bottom, after a week of application of the preparation, the defect is pure granulated, without secretion, with a quiet environment.

4) Male, 77 years old, with a puncture after lowering the 2nd, 3rd and 4th toes for diabetic gangrene, size 5x4 cm, depth up to 3 cm 8 with abundant purulent secretion and extensive adhering necroses throughout the wound range. After 4 weeks of application of the product in 1/5 of the defect, clean granulations were formed, in the other part the necrotic process was not limited due to poor blood circulation in the periphery of the foot in microangiopathy and obliterating arteriosclerosis.

5) Man, 56 years old, with a defect after incision, absent phlegmons of the intercouple joint of the toe, extending intraarticularly, 2.5 cm in diameter, with necroses and purulent secretion - after a weekly application of the product, retreat the secretion, clean the defect. clean granulation, the defect surroundings calmed down.

6) female, 78 years old, with a defect after amputation of the 1st and 2nd toes including the head of the appropriate metars for diabetic gangrene, size 5x3 cm, with a covered bottom, adhering necroses and purulent secretion in the distal pole of the wound. 4 weeks of application achieved clean granulations throughout the wound range, suppressed purulent secretion and continued epithelialization to reduce the defect to 3 x 2 cm.

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Žena) woman, ΊΊ years old, with 2 defects in the amputation stump of the lower leg after amputation for diabetic gangrene followed by absenting stump phlegm, who made it possible to dissolve the wound in its entirety - defects of 10 x 2-4 cm, with purulent secretion, coated granulation and extensive necroses, adhering firmly to the bottom. After 4 weeks of application, the granules were cleaned, necrosis released, secretion retreated and continued epithelialization from the edges of the defects so that they were reduced meddally to 3 cm in diameter, later to 1.5 cm in diameter.

Claims (2)

SUBJECT OF THE INVENTION CLAIMS 1. Proteolytic wound cover, characterized in that it consists of spherical particles with a diameter of 0.05 to 0.5 mm, preferably 0.1 to 0.3 mm, based on Uerivatized cellulose with immobilized enzymes of the family of chymotrypsin, trypsin, subtilisin. 2. A method according to claim 1, characterized in that the regenerated pearled regenerated cellulose in water, never dried, with a particle size of 0.07 to 0.7 mm, preferably 0.14 to 0.4 mm, is completely freed by washing, possibly by steam distillation, of toxic impurities, activated for enzyme binding, it is an isolation of proteases from the group of chymoZrypuin, trypsin, subtisils, alternately washed with a buffer of pH 8.5 to 9.5, and a buffer of pH 4-5 to zero the proteolytic activity of the wash liquid, then washed with a buffer of pH 7.5-8.5 and in this medium dried to 0.1-15% residual water content in the dry matter, preferably by lyophilization.